Glycerol oxidase and process for the production thereof and method for the quantitative determination of glycerol by glycerol oxidase

ABSTRACT

Glycerol oxidase is an oxidizing enzyme of glycerol characterized by its ability to oxidize glycerol in the presence of oxygen to form hydrogen peroxide and glyceraldehyde. This enzyme is produced by cultivation of a microorganism belonging to the genus Aspergillus or the genus Neurospora in a nutrient medium.

This is a division of application Ser. No. 897,695, filed Apr. 19, 1978now U.S. Pat. No. 4,202,941, issued May 13, 1980.

This invention relates to an oxidizing enzyme of glycerol, which will behereinafter referred to as "glycerol oxidase", a process for producingthis oxidizing enzyme, and a novel method for the quantitativedetermination of glycerol by the use of the glycerol oxidase.

In accordance with the present invention, a novel enzyme, glyceroloxidase, and a novel method for the quantitative determination ofglycerol by said enzyme are first provided.

The present inventors have made various studies of enzymes using variousmicroorganisms belonging to the genus Aspergillus and the genusNeurospora. As a result, the inventors have found that there is anenzyme in the culture broth obtained by culturing microorganisms, suchas Aspergillus joponicus KY-45; Neurospora crassa KY-462, etc. Thepresent inventors have investigated properties of the enzyme, have foundthat the enzyme is a new enzyme which selectively oxidizes glycerol, andhave named the enzyme "glycerol oxidase".

Heretofore, such a new enzyme has been in demand but its presence hasnot been confirmed.

The novel enzyme provided according to the process of the presentinvention has the following enzymological and physicochemicalproperties:

(1) Action:

The present glycerol oxidase oxidizes glycerol under the consumption ofoxygen to form hydrogen peroxide and glyceraldehyde.

(2) Substrate specificity:

The present enzyme reacts very specifically with glycerol.

(3) Optimum pH and stable pH range:

(3.1) Optimum pH;

Glycerol oxidase produced by the microorganisms of the genus Aspergillus(which will be hereinafter referred to as "enzyme of the genusAspergillus"): pH 7-8

Glycerol oxidase produced by the microorganisms of the genus Neurospora(which will be hereinafter referred to as "enzyme of the genusNeurospora"): pH 8.0-8.5

(3.2) Stable pH range:

Enzyme of the genus Aspergillus: pH 5.0-8.0

Enzyme of the genus Neurospora: pH 5.0-8.0

(4) Method for the determination of the enzyme activity:

The enzyme activity is expressed by "unit." A "unit" of the enzymeactivity is defined as that amount of enzyme which will decompose 1μmole of glycerol at 37° C. in 1 minute in the presence of oxygen. Thedetermination of the enzyme activity is carried out as follows: glycerolis reacted with glycerol oxidase with stirring to form hydrogenperoxide, and 4-aminoantipyrine and phenol are reacted with theresulting hydrogen in the presence of peroxidase to derive quinoneiminepigment. The optical density at 500 nm of the resulting uinoneiminepigment is measured to determine the amount of generated hydrogenperoxide, whereby the enzyme activity is determined (which will behereinafter referred to as "4-AA method").

In the present invention, specific activity is defined by unit per 1 mgof protein. The amount of the enzyme protein is measured according toLowry method using copper-Folin reagent [O. H. Lowry, N.J. Rosebrough,A. L. Farr and R. J. Randall: J. Biol. Chem., 193, 265 (1951)].

(5) Optimum temperature range for action:

Enzyme of the genus Aspergillus: about 40° C.

Enzyme of the genus Neurospora: 40°-45° C.

(6) Conditions for Inactivation by pH, temperature, etc.:

(6.1) Condition for Inactivation by temperature:

Enzyme of the genus Aspergillus is inactivated about 60% after treatmentat 45° C. for 30 minutes and substantially inactivated after treatmentat 60° C. for 30 minutes.

Enzyme of the genus Neurospora is inactivated about 50% after treatmentat 25° C. for 30 minutes and substantially inactivated after treatmentat 50° C. for 30 minutes.

(6.2) Condition for Inactivation by pH:

Enzyme of the Aspergillus is inactivated at a pH value of above 10 orbelow 4 for 30 minutes.

Enzyme of the genus Neurospora is inactivated at a pH the same as aboveindicated.

(7) Inhibition, activation and stabilization:

(7.1) Inhibitor:

Enzymes activities obtained by adding 1 mM of various inhibitors areshown in Table 1, the activity of the enzyme with no addition of theinhibitor being defined as 100. The activities are measured according to4-AA method deriving a pigment of 4-aminoantipyrine system from thegenerated H₂ O₂.

                  TABLE 1                                                         ______________________________________                                                           Enzyme                                                                          Enzyme of Enzyme of                                                           the genus the genus                                      Inhibitor            Aspergillus                                                                             Neurospora                                     ______________________________________                                        None                 100       100                                            AgNO.sub.3           30        --                                             HgCl.sub.2           20        23                                             Pb(CH.sub.3 COO).sub.2                                                                             15        --                                             CuSO.sub.4           10        20                                             ZnSO.sub.4           --        23                                             FeSO.sub.4           --        0                                              NaN.sub.3            20        --                                             0-phenanthroline     90        --                                             α,α'-dipyridyl                                                                         85        --                                             PCMB (p-chloromercuribenzoate)                                                                     50        108                                            N-ethylmaleimide     100       --                                             NH.sub.2 OH--HC1      0         0                                             8-hydroxyquinoline   90        --                                             diethyldithiocarbamate                                                                             35        20                                             neocuproine          100       --                                             iodoacetate          --        100                                            cysteine             --        60                                             dithiothreitol       100       --                                             EDTA                 105       80                                             ______________________________________                                    

(7.2) Activation:

No compound has been found yet especially for the activation.

(7.3) Stabilization:

Enzyme of the genus Aspergillus can be stabilized by adding 0.1 mM to1.0 mM of a sulfhydryl compound such as mercaptoethanol, dithiothreitol,etc. thereto.

(8) Method for purification:

Purification can be carried out according to the following conventionalprocedures: (1) fractional precipitation by ammonium sulfate, (2) columnchromatography on DEAE cellulose, (3) sieving fractionation by Sephadex,(4) column chromatography on hydroxyapatite, (5) freeze-drying of activefractions, etc. as will be hereinafter described in detail.

(9) Molecular weight:

The molecular weight of the present enzyme is determined according tothe elution pattern by analysis using Sephadex G-200 column. As theresult, the enzyme of the genus Aspergillus is determined to have amolecular weight of 300,000 or more.

(10) Crystal structure and elemental analysis:

No crystallization has been succeeded yet, and no determination has beenmade yet.

It has been clarified that the enzyme provided according to the presentinvention is a novel enzyme having the foregoing properties, that is,glycerol oxidase.

Now, a novel process for producing the enzyme and a process forpurifying the same provided according to the present invention will bedescribed in detail below.

Glycerol oxidase can be obtained by culturing a microorganism having anability to produce glycerol oxidase and belonging to the genusAspergillus or the genus Neurospora in a nutrient medium containing anappropriate carbon source, nitrogen source, inorganic materials andother nutrients, and recovering glycerol oxidase from the resultingculture broth.

Any microorganism can be used, so long as it belongs to the genusAspergillus or the genus Neurospora, and has an ability to produceglycerol oxidase. As suitable microorganisms, Aspergillus japonicusKY-45 (FERM-P No. 3959, NRRL 11102), Aspergillus oryzae KY-63 (NRRL11103), Aspergillus parasiticus KY-77 (NRRL 11104), Aspergillus flavusKY-98 (NRRL 11105), Neurospora crassa KY-462 (FERM-P NO. 3960, NRRL11106), Neurospora sitophila KY-445 (NRRL 11264), Neurospora tetraspermaKY-447 (NRRL 11265), etc. can be mentioned.

Microbiological properties of these microorganisms are disclosed in thefollowing literatures:

    ______________________________________                                        Aspergillus japonicus:                                                                      The genus Aspergillus: The Williams                                           & Wilkins Co., Baltimore, 1965,                                               P. 327-328                                                      Aspergillus oryzae:                                                                         ibid, p. 370-373                                                Aspergillus parasiticus:                                                                    ibid, p. 369-371                                                Aspergillus flavus:                                                                         ibid, p. 361-365                                                Neurospora crassa:                                                                          Comparative Morphology of Fungi,                                              McGraw-Hill Book Company, Inc.,                                               New York and London 1928, p. 227;                                             Jour. Agr. Res., 34, p. 1019-1042 (1927)                        Neurospora sitophila:                                                                       ibid, p. 226-227                                                Neurospora tetrasperma:                                                                     ibid, p. 227                                                    ______________________________________                                    

Any of synthetic medium and natural medium can be used as a medium inthe present invention, so long as it properly contains a carbon source,nitrogen source, inorganic materials and other nutrients.

As the carbon source, carbohydrates such as glucose, blackstrapmolasses, etc., and sugar alcohols such as glycerol, sorbitol, mannitol,etc. can be used.

As the nitrogen source, ammonia, various inorganic and organic ammoniumcompounds such as ammonium chloride, ammonium sulfate, ammoniumcarbonate, ammonium phosphate, ammonium acetate, etc., nitrogencompounds such as urea, etc., nitrogenous organic materials such aspeptone, yeast extract, casein hydrolyzate, defatted soybeans, or thedigested products thereof, etc., can be used.

As the inorganic materials, salts of such metals as sodium, potassium,manganese, magnesium, calcium, cobalt, nickel, zinc, copper, etc., andsalts of chromium, sulfuric acid, phosphoric acid, nitric acid,hydrochloric acid, etc., can be used.

In the present invention, glycerol oxidase can be obtained in bestyield, when a glycerol oxidase producing microorganism is cultured in amedium containing glycerol. For example, productivity of glyceroloxidase in terms of activity obtained when the microorganism is culturedin a medium containing glycerol is 10 to 100 times as high as thatobtained when the microorganism is cultured in a medium containing 1g/dl glucose, 1 g/dl malt extract and 0.5 g/dl yeast extract or theCzapeck medium.

It is preferable that 0.1 to 5 g/dl of glycerol is added to a medium.

Culturing is usually carried out at a temperature of 20° to 40° C.,preferably 25° to 35° C., and at a pH of 6 to 8 at the start ofculturing, preferably at about 7. A considerable amount of glyceroloxidase is formed in the resulting culture broth by shaking culture oraeration-agitation submerged culture under said conditions for 30 to 72hours.

Glycerol oxidase thus formed in the resulting culture broth can berecovered in the following manner:

Since glycerol oxidase is usually formed in the microbial cells, theprocedure for recovering the enzyme from the microbial cells isdescribed below. Microbial cells obtained by filtration orcentrifugation of culture broth after the completion of culturing aresufficiently washed with water or buffer solution. Then, the microbialcells are suspended in an appropriate amount of a buffer solution, anddisrupted. Disruption is carried out by mechanical disintegration [forexample, by mortar, Dyno-mill (product of Willy A. Bachofen,Switzerland), Manton goulin, French press, Fuse press, ultrasonicdisintegrator, etc.].

Solid mterials are removed from the resulting solution of the disruptedmicrobial cells by filtration or centrifugation, and then glyceroloxidase is recovered from the solution according to the conventionalprocedure for isolating enzymes. For example, enzyme powders can beobtained according to steps of (1) fractional precipitation withammonium sulfate, (2) column chromatography on DEAE cellulose, (3) sievefractionation by Sephadex, (4) column chromatography on hydroxyapatite,and (5) freeze-drying of active fractions. Of course, repetition of thesame operating steps, and other conventional purification methods can beused in combination, if required.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amount of oxygen uptake in a reaction system whereglycerol oxidase is reacted with glycerol in the presence of oxygen. Inthis figure, curve A represents the amount of the oxygen uptake in thereaction system free from catalase and curve B the amount of the oxygenuptake in the presence of catalase.

FIG. 2 shows relative activities of the enzyme of the genus Aspergillusafter incubation at 37° C. and various pH for 10 minutes.

FIG. 3 shows residual activities of the enzyme of the genus Aspergillusafter treatment at 30° C. and various pH for 30 minutes.

FIG. 4 shows relative activities of the enzyme of the genus Neurosporaafter incubation at 37° C. and various pH for 10 minutes.

FIG. 5 shows residual activities of the enzyme of the genus Neurosporaafter treatment at 30° C. and various pH for 30 minutes.

FIG. 6 shows relation between reaction time and OD value (at 500 nm) inmeasuring the activity of the enzyme.

FIG. 7 shows the enzyme activities of the enzyme of the genusAspergillus after incubation at pH 8 and various temperatures for 10minutes.

FIG. 8 shows the enzyme activities of the enzyme of the genus Neurosporaafter incubation at pH 8 and various temperatures for 10 minutes.

FIG. 9 shows residual activities of the enzyme of the genus Aspergillusafter treatment at pH 7 and various temperatures in 0.1 M ammoniumbuffer solution for 30 minutes.

FIG. 10 shows residual activities of the enzyme of the genus Neurosporaafter treatment at pH 7 and various temperatures in 0.1 M ammoniumbuffer solution for 30 minutes.

FIG. 11 shows relation between substrate (glycerol) concentration andoptical density of reaction solutions at 500 nm in Table 4.

Enzymological and physico-chemical properties of glycerol oxidase thusobtained are described below (as glycerol oxidase, the respectivepurified preparates obtained in Examples 1 and 2, are used).

I. Action

The enzyme specifically oxidizes glycerol under the consumption ofoxygen to form hydrogen peroxide and glyceraldehyde.

(a) Confirmation of formation of hydrogen peroxide:

(a)-1. Glycerol oxidase is reacted with glycerol in the presence ofoxygen, and then peroxidase, phenol and 4-aminoantipyrine are added tothe enzyme system, whereby quinoneimine pigment is formed in thereaction system [reaction of hydrogen peroxide with peroxidase, phenoland 4-aminoantipyrine is disclosed in Clin. Chem. 20, p. 470 (1974)].

(a)-2. Glycerol oxidase is reacted with glycerol in the presence ofoxygen to form hydrogen peroxide, and catalase is added to decompose thegenerated hydrogen peroxide. Then, peroxidase, phenol and4-aminoantipyrine are added to the reaction system to conduct the samereaction as above, but the same quinoneimine pigment is not formed inthe reaction system.

(a)-3. When catalase is presented in the glycerol oxidation systemcatalyzed by glycerol oxidase in the presence of oxygen, the amount ofoxygen uptake is decreased to half. This fact is supported by thefollowing experimental facts:

(a)-3-1. Reaction composition A (system free from catalase):

    ______________________________________                                        Reagents                                                                      ______________________________________                                        Aqueous 0.01M glycerol solution                                                                        0.5 ml                                               0.1M NH.sub.4 OH-NH.sub.4 Cl buffer solution                                  (which will be hereinafter referred                                           to as "ammonium buffer solution")                                             (pH 8.0)                 1.0 ml                                               Aqueous glycerol oxidase solution                                                                      0.2 ml *1                                            Aqueous 2.4 mM 4-aminoantipyrine solution                                                              0.5 ml                                               Aqueous 42 mM phenol solution                                                                          0.5 ml                                               Aqueous peroxidase solution                                                                            0.2 ml *2                                            Water                    0.1 ml                                               ______________________________________                                         *1 5 μ moles of glycerol oxidase having a specific activity of 30          obtained in Example 1 of the present invention.                               *2 Made by Sigma Corp. (U.S.A.), containing 200 units of specific activit     of 1,000.                                                                

(a)-3-2. Reaction Composition B (system containing catalase):

    ______________________________________                                        Reagents                                                                      ______________________________________                                        Aqueous 0.01M glycerol solution                                                                       0.5 ml                                                0.1M ammonium buffer solution (pH 8.0)                                                                1.0 ml                                                Aqueous glycerol oxidase solution                                                                     0.2 ml *1                                             Aqueous catalase solution                                                                             1.0 ml *3                                             Water                   0.3 ml                                                ______________________________________                                         *1 Same as in Reaction Composition A                                          *3 Made by Sigma Corp. (U.S.A.), containing 100 units of catalase having      specific activity of 100.                                                

(a)-3-3. Reaction operation:

In case of the system free from catalase, the reagents shown in ReactionComposition A are mixed, and reaction is carried out at 37° C. withstirring. The amount of oxygen uptake is measured by a Warburg'smanometer. The results are shown in FIG. 1.

In case of the system containing catalase, the reagents shown inReaction Composition B are mixed, and reaction is carried out at 37° C.Similarly, the amount of oxygen uptake is measured, and is shown inFIG. 1. As is evident from FIG. 1, 4.95μ moles of oxygen is consumed inthe absence of catalase (curve A in FIG. 1) for 5.0μ moles of glycerolas a substrate.

In the presence of catalase (curve B in FIG. 1), 2.49μ moles of oxygenis consumed, which is equal to about one-half of that of (A).

It is confirmed from the foregoing reaction systems (a)-1, (a)-2 and(a)-3 that the present enzyme can form hydrogen peroxide.

Quantitative determination of the generated hydrogen peroxide is made bycolorimetric quantitative determination of the formed quinoneiminepigment. That is, it is confirmed by the quantitative determination ofthe formed hydrogen peroxide in the system of Reaction Composition Aaccording to 4-AA method that 4.92μ moles of hydrogen peroxide is formedfrom 5μ moles of glycerol.

(b) Confirmation of formation of glyceraldehyde:

(b)-1. Glycerol oxidase is reacted with glycerol in the presence ofoxygen, and a reaction product is identified to be glyceraldehyde.

(b)-1-1. Procedure for identification:

(b)-1-1-(1). Reaction solution:

Five drops of an aqueous solution containing 50 mg/ml of glycerol, 5drops of solution containing 10 u/ml of glycerol oxidase in 0.02 MTris-HCl buffer solution, and 1 drop of 14,000 u/ml of catalase arecollected, and reaction is carried out at 30° C. with shaking for 20hours.

(b)-1-1-(2). Identification:

As a result of the thin layer chromatography given below (which will behereinafter referred to as "TLC"), the product in the reaction solutionis identified as glyceraldehyde by comparison with the authenticsubstance.

The thin layer plate used is silica gel G-60F-254 (trademark of E.Merck, West Germany), and developing solvents are solvent system 1[butanol:acetic acid:water=4:1:1 (by volume)], and solvent system 2[butanol:pyridine:water=3:2:1.5 (by volume)].

After the development, three kinds of reactions, that is, p-anisidinehydrochloric acid reaction, periodic acid-benzidine reaction, and2,4-dinitrophenylhydrazine reaction are conducted on the plate, and itis confirmed that Rf values of the reaction products and color tones ofthe spots formed by said reactions are identical with those of theauthentic substances. The results are given in Table 2.

                  TABLE 2                                                         ______________________________________                                        Identification of reaction products by TLC                                    (Rf values and coloring reaction)                                             coloring                                                                              Developing solvent          Solvent                                   agent   sample         Solvent System 1                                                                           system 2                                  ______________________________________                                        P-                                                                            anisidine                                                                     hydro-  Glyceraldehyde 0.67         0.76                                      chloric (authentic substance)                                                                        Rf value (brown)                                                                           (brown)                                   acid    Reaction product                                                                             Rf value 0.67                                                                              0.77                                      reagent.sup.1          (brown)      (brown)                                   Periodic                                                                              Glyceraldehyde 0.66         0.76                                      acid-   (authentic substance)                                                                        Rf value (+)*                                                                              (+)*                                      benzidine              0.67         0.77                                      reagent.sup.2                                                                         Reaction product                                                                             Rf value (+)*                                                                              (+)*                                      2,4-                                                                          dinitro-                                                                              Glyceraldehyde 0.67         0.77                                      phenyl- (authentic substance)                                                                        Rf value (orange)                                                                          (orange)                                  hydrazine                                                                             Reaction product                                                                             Rf value 0.68                                                                              0.77                                      reagent.sup.3          (orange)     (orange)                                  ______________________________________                                         Color indicated in parentheses shows color tone of the spot, and (+)* in      periodic acidbenzidine reagent means white spot in blue background.           Notes                                                                         .sup.1 panisidine hydrochloric acid reagent: reductive carbonyl compounds     such as sugars react with panisidine hydrochloric acid to exhibit             coloration characteristic of respective structures.                           .sup.2 periodic acidbenzidine reagent: polyalcohol and compounds having a     similar structure thereto consume periodic acid. Thus, those compounds ar     detected by a white spot.                                                     .sup.3 2,4dinitrophenylhydrazine reagent: carbonyl compound reacts with       2,4dinitrophenylhydrazine to form 2,4dinitrophenylhydrazone, and is           detected by an orange spot.                                              

As shown in the foregoing table, the products and the glyceraldehydeauthentic substance are completely identical with one another in the Rfvalues and coloration by the coloring reagents. Thus, it is confirmedthat the product obtained by reaction of glycerol oxidase with glycerolin the presence of oxygen is glyceraldehyde.

(b)-2. The amount of glyceraldehyde formed when glycerol oxidase isreacted with glycerol in the presence of oxygen is almost equimolar tothat of consumed glycerol. The fact is confirmed by adding2,4-dinitrophenylhydrazine to the reaction system to prepare2,4-dinitrophenylhydrazone of the product and quantitatively determiningthe latter product by colorimetry.

(c) Confirmation of the amount of oxygen uptake:

Consumption of oxygen in the system where glycerol oxidase is reactedwith glycerol is measured by an oxygen electrode and a Warburg'smanometer. As a result, it is confirmed that the amount of oxygen uptakemeets with the amount of the formed glyceraldehyde.

(d) Quantitative determination of the amount of hydrogen peroxide, theamount of glyceraldehyde and the amount of oxygen uptake are carried outaccording to the procedures described in the above three items (a), (b)and (c). As a result, it is found that those obtained data arestoichiometrically reasonable.

It is confirmed from said qualitative and quantitative experimentalresults that the present enzyme oxidizes glycerol to form hydrogenperoxide and glyceraldehyde according to the following equation, thatis, that the present enzyme is glycerol oxidase. ##STR1##

II. Substrate specificity

The relative activity is measured using other substrates in equimolaramounts to that of glycerol in the procedure for measuring the activityaccording to 4-AA method.

The relative activities on other substrates are given in Table 3, theactivity on glycerol being defined as 100.

                  TABLE 3                                                         ______________________________________                                                         Enzyme of   Enzyme of                                        Enzyme           the genus   the genus                                        Substrate        Aspergillus Neurospora                                       ______________________________________                                        glycerol         100         100                                              1,2-propanediol  10.5        0                                                1,3-propanediol  3.3         0                                                1,3-butanediol   2.0         0                                                Glycerol-3-phosphoric acid                                                                     0.4         21                                               1,4-butanediol   0.3         0                                                2,3-butanediol   0.3         0                                                ethanol          0           0                                                n-propyl alcohol 0           0                                                isopropyl alcohol                                                                              0           0                                                ______________________________________                                    

III. Optimum pH and stable pH range

1. Optimum pH of enzyme of the genus Aspergillus is in a range of 7 to8.

The activities are measured after incubation at 37° C. at various pHvalues for 10 minutes. The results are shown in FIG. 2.

2. Stable pH range of enzyme of the genus Aspergillus is 5.0 to 8.0.

Residual activities are measured after treatment at 30° C. and variouspH for 30 minutes, using the following buffer solutions. The results areshown in FIG. 3.

Note:

pH 4-5: Acetate buffer solution is used.

pH 6-7: Tris-HCl buffer solution is used.

pH 8-9: Borate buffer solution is used.

3. Optimum pH of enzyme of the genus Neurospora is 8.0 to 8.5.

Activity is measured after incubation at 37° C. and various pH for 10minutes. The results are shown in FIG. 4.

4. Stable pH range of enzyme of the genus Neurospora is 5 to 8.0.Measurement is carried out in the same manner as in the foregoing itemIII-2. The results are shown in FIG. 5.

IV. Procedure for determination of the enzyme activity

(a) Principle:

Determination of the enzyme activity is carried out by reacting hydrogenperoxide generated by the enzyme with 4-aminoantipyrine and phenol inthe presence of peroxidase to obtain quinoneimine pigment, andquantitatively determining the resulting quinoneimine pigment.

Reaction formulae are given by the following equations (1) and (2):##STR2##

Reaction principle of equation (2) is disclosed by C. C. Allain et al.:Clin Chem. 20, 470 (1974).

(b) Reagents:

    ______________________________________                                        (1)   Substrate: aqueous 0.1M glycerol                                              solution                 0.5 ml                                         (2)   Buffer solution: 0.1M ammonium buffer                                         solution (pH 8)          1.0 ml                                         (3)   4-Aminoantipyrine: aqueous 2.4 mM                                             solution                 0.5 ml                                         (4)   Phenol: 42 mM (aqueous solution)                                                                       0.5 ml                                         (5)   Aqueous peroxidase solution:                                                                           0.1 ml                                               (Amount of protein: 2 mg/ml,                                                  Specific activity: 100)                                                 (6)   Water                    0.3 ml                                         (7)   Aqueous enzyme solution  0.1 ml                                         ______________________________________                                    

(c) Operational procedure:

The reagents (1)-(6) are mixed in a test tube, and then shaken at 37° C.for 5 minutes. Then, enzyme solution is added thereto, and thus obtainedmixture is made up to 3 ml with ammonium buffer solution. Reaction iscarried out at 37° C. for 10 minutes with shaking. On the other hand,similar procedure is repeated using water in place of the test solutionas a reference. The optical density of the reaction solution at 500 mmis measured, and a difference from the control is determined as ΔOD(optical density).

(d) Calculation of the enzyme activity:

One unit of glycerol oxidase is the amount of the enzyme which willdecompose 1μ mole of glycerol at 37° C. in 1 minute.

On the other hand, the absorption coefficient of 0.5 mM of quinoneiminepigment is reported as 5.33 [Clin Chem. 20, 470 (1974)], and thus, ifthe optical density (ΔOD) at 500 nm of 3 ml of the reaction solutionobtained according to the foregoing operational procedure IV-(c) isrepresented by a, the desired enzyme activity (A) per milliliter of theenzyme solution is calculated from the following formula: ##EQU1##

OD values at 500 nm of reaction solutions are measured by changing thereaction time in the measurement of the enzyme activity, and the resultsshown in FIG. 6 are obtained.

It is evident from FIG. 6 that the OD values at 500 nm are proportionalto the reaction times.

V. Optimum temperature range for action

(1) Enzyme of the genus Aspergillus:

The enzyme activities after incubation at pH 8 and various temperaturesfor 10 minutes are shown in FIG. 7. The optimum temperature is at about40° C.

(2) Enzyme of the genus Neurospora:

The enzyme activities after incubation at pH 8 and various temperaturesfor 10 minutes are shown in FIG. 8. The optimum temperature is at about35° to 45° C.

VI. Inactivation conditions by pH, temperature, etc.

Inactivation by pH condition:

As described in the foregoing item III on the optimum pH and stable pHrange, the enzyme of the genus Aspergillus is substantially 100%inactivated below pH 4 or above pH 10. Similarly, the enzyme of thegenus Neurospora is substantially 100% inactivated below pH 4 or abovepH 10.

Inactivation by temperature:

Residual activity is measured after heat treatment at pH 7.0 for 30minutes in 0.1 M Tris-HCl buffer solution.

The results as to the enzyme of the genus Aspergillus are shown in FIG.9, and those as to the enzyme of the genus Neurospora in FIG. 10. Theformer is 100% stable up to 40° C., but is about 60% inactivated at 45°C. The latter is about 50% inactivated at 30° C. and about 100%inactivated at 50° C. The former (the enzyme of the genus Aspergillus)is stabilized by addition of 0.1 mM dithiothreitol, and its thermalresistance is also increased (see FIG. 9 where it is stable up to about50° C.).

Now, a method for the quantitative determination of glycerol by thenovel enzyme, glycerol oxidase, provided according to the presentinvention is described below.

The following methods are available for the quantitative determinationof glycerol:

(a) A method which comprises reacting glycerol oxidase with glycerol inthe presence of oxygen, and quantitatively determining the formedhydrogen peroxide.

(b) A method which comprises reacting glyceraldehyde which is formed inthe above (a) with 2,4-dinitrophenylhydrazine, forming2,4-dinitrophenylhydrazone, quantitatively determining the2,4-dinitrophenylhydrazone by colorimetry, and thereby quantitativelydetermining glycerol.

(c) A method which comprises reacting glycerol oxidase with glycerol inthe presence of oxygen, and measuring an amount of oxygen uptake of thesystem.

The principles and the procedures of the methods (a), (b) and (c) aredescribed in the above item I. However, as an example, the method (a)for the quantitative determination of glycerol by measuring the amountof the formed hydrogen peroxide will be described below:

The optical density of reaction solutions at 500 nm is determinedaccording to the operational procedure described in the foregoing itemIV-(c), using solutions containing 0.1 mg of the enzyme having aspecific activity of 3.2 per milliliter of the solutions changing theconcentration of the substrate (glycerol) solution to 0.1 mM, 0.2 mM,0.5 mM, 1.0 mM, 5.0 mM, and 10.0 mM in the foregoing item IV-(b). Theresults are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                         Substrate                                                                    cocen-                                                                        tration                                                                       OD value                                                                              0.1 mM  0.2mM   0.5mM 1.0mM 5.0mM 10.0mM                              ______________________________________                                        0D value                                                                      at 500 nm                                                                             0.060   0.121   0.303 0.605 3.002 5.998                               ______________________________________                                    

It is observed that there is a linear relationship between the substrateconcentration (glycerol concentration) and the OD value of reactionsolutions at 500 nm. On the basis of this principle, glycerol containedin solution at an unknown concentration can be quantitativelydetermined.

Thus, the glycerol concentration of a solution can be measured usingglycerol oxidase. This fact will suggest a new procedure and kit for thequantitative determination of glycerol or derivatives thereof.

A method for the quantitative determination of glycerol has been indemand, for example, in the field of biochemistry. Various methods areknown for the quantitative determination of triglyceride by hydrolyzingtriglyceride in serum to form glycerol and fatty acid, and measuring theglycerol.

As the chemical method for the quantitative determination of glycerol,chromotropic acid method, acetylacetone method, triazole method, Randrupmethod, and Mendelsohn fluorescense method are known, but all thosemethods have such a disadvantage that the reactions in those methods arenon-specific to glycerol.

As the enzyme method, a method using glycerokinase (E.c.2.7.1.30) isknown, but the reaction must be conducted together with pyruvate kinase(E.c.2.7.1.40) and lactate dehydrogenase (E.c.1.1.1.27), and thus ittakes much time in the measurement and consequently the method isunsuitable for the treatment of a large number of samples.

It has been found that the present glycerol oxidase directly oxidizesglycerol to form hydrogen peroxide stoichiometrically. Thus, theobtained hydrogen peroxide is easily converted to a coloring system, andquantitative determination of glycerol, that is, quantitativedetermination of triglyceride can be very simply and specifically madeby the colorimetrical quantitative determination.

Now, the present invention will be described in detail below, referringto examples.

EXAMPLE 1

Aspergillus japonicus KY-45 (ATCC 1042, NRRL 11102, FERM-P No. 3959) isinoculated in 300 ml of seed medium containing 10 g/l of glycerol, 10g/l of malt extract, and 5 g/l of yeast extract (pH 6.2 beforesterilization) in a 2-l Erlenmeyer flask, and cultured at 30° C. withshaking for 48 hours. Nine hundred milliliters (corresponding to threeflasks) of the resulting seed culture is inoculated in 15 l of the samemedium as said seed medium in a 30-l jar fermenter, and cultured at 30°C. with aeration (15 l/min.) and stirring (250 rpm) for 40 hours. Afterthe culturing, the resulting culture broth is filtered through a Buchnerfunnel, and the cakes are washed with water, whereby about 150 g (drybasis) of microbial cells is obtained.

The microbial cells are suspended in 5 l of 10 mM ammonium buffersolution (pH 8.0), and disrupted in Dyno-mill (made by Willy A.Bachofen, Switzerland). After the disruption, the disrupted suspensionis centrifuged by a freezing centrifuge (20,000×G for 20 minutes), and4.7 l of supernatant (protein content: 51 g, specific activity: 0.05units/mg) is obtained. The resulting supernatant is admixed withammonium sulfate, and fractions precipitated at 30-70% saturation withammonium sulfate are collected, and dissolved in 50 ml of 10 mM ammoniumbuffer solution (pH 8.0). The resulting solution is dialyzed against 10l of 10 mM ammonium buffer solution, using a cellophane tube as adialysis membrane.

Dialysis is continued for 48 hours with total of 40 l of dialysissolution, while exchanging the dialysis solution at every 12 hours.Dialyzed enzyme solution is passed through a column (5.5×40 cm) of DEAEcellulose (made by Serva Co., West Germany) equilibrated in advance with10 mM ammonium buffer solution (pH 8.0). Enzyme is adsorbed through saidoperation, and unadsorbed impure protein is washed out with the samebuffer. Then, the elution is carried out by a linear gradient of 0.1 Mto 0.2 M ammonium sulfate in 2 l of 10 mM ammonium buffer solution (pH8.0), whereby glycerol oxidase is eluted. Five hundred milliliters ofthe active fractions (protein content: 1.2 g, specific activity 1.1) arecollected, and admixed with ammonium sulfate to make 70% saturation andto precipitate the enzyme.

Then, precipitates are collected by centrifugation (20,000×G for 20minutes), and dissolved in 50 ml of 10 mM ammonium buffer solution (pH8.0). The solution is passed through a column (5.5×80 cm) of SephadexG-100 (made by Pharmacia Fine Chemicals, Co., Sweden) equilibrated inadvance with 10 mM ammonium buffer solution (pH 8.0). One liter of 10 mMammonium buffer solution (pH 8.0) is passed through the column andeluate is obtained in fractions. From the fractions, 300 ml of afraction having a high specific activity (protein content: 310 mg,specific activity: 3.2) is obtained, and admixed with ammonium sulfateto make 70% saturation and to precipitate enzyme. Then, the precipitatesare collected by centrifugation (20,000×G for 20 minutes) and dissolvedin 20 ml of 10 mM ammonium buffer solution (pH 8.0). The resultingsolution is dialyzed for 24 hours against 5 l of 10 mM ammonium buffersolution (pH 8.0), using a cellophane tube as a dialysis membrane, whiletwice exchanging the dialysis solution. After the dialysis, theresulting enzyme solution is passed through a column (5.5×20 cm) ofhydroxyapatite equilibrated in advance with 10 mM ammonium buffersolution (pH 8.0). Furthermore, 250 ml of the same buffer is passedthrough the column, and eluate is obtained in fractions. Fractionshaving a specific activity higher than 20 are collected, andfreeze-dried, whereby 10.2 mg of purified powdery enzyme preparate ofglycerol oxidase (specific activity: 30.2) is obtained. The purifiedenzyme has a specific activity about 610 times as high as that of cellextract. The yield is 19% in terms of activity.

EXAMPLE 2

In place of Aspergillus japonicus KY-45 of Example 1, Neurospora crassaKY-462 (NRRL 11106, FERM-P No. 3960) is inoculated in five 2-lErlenmeyer flasks containing 10 g/l of glycerol, 10 g/l of malt extract,and 5 g/l of yeast extract and cultured at 30° C. for 48 hours withshaking in the same manner as in Example 1.

The resulting culture broth is extracted and purified in the same manneras in Example 1, whereby 1.1 mg of purified enzyme preparate of glyceroloxidase (specific activity: 14.1) is obtained in a 10.5% yield in termsof activity.

EXAMPLE 3

In this example, the procedure in Example 1 is repeated except that fivestrains shown in Table 5 are used in place of Aspergillus japonicusKY-45. The glycerol oxidase activities of the resulted supernatant ofcell extract are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                         Strains                 Activity u/l                                         ______________________________________                                        Aspergillus oryzae KY 63 (NRRL 11103)                                                                  320                                                  Aspergillus parasticus KY 77 (NRRL 11104)                                                              216                                                  Aspergillus flavus KY 98 (NRRL 11105)                                                                  108                                                  Neurospora sitophila KY 445 (NRRL 11264)                                                               325                                                  Neurospora tetrasperma KY 447 (NRRL 11265)                                                             105                                                  ______________________________________                                    

EXAMPLE 4

(a) Reagents to be used:

    ______________________________________                                        (1)   Test solution: aqueous glycerol                                               solution (concentration: unknown)                                                                    0.5 ml                                           (2)   Buffer solution: 0.1M ammonium                                                buffer solution (pH 8) 1.0 ml                                           (3)   4-aminoantipyrine: aqueous 2.4 mM                                             solution               0.5 ml                                           (4)   Phenol: aqueous 42 mM solution                                                                       0.5 ml                                           (5)   Peroxidase: aqueous peroxidase                                                solution (protein content: 20 mg/ml,                                          specific activity: 1000)                                                                             0.1 ml                                           (6)   Water                  0.3 ml                                           (7)   Enzyme: Glycerol oxidase obtained                                             in Example 1 (protein content:                                                1.0 mg/ml, specific activity: 3.2)                                                                   0.1 ml                                           ______________________________________                                    

(b) Procedure:

The above reagents (1)-(6) are placed in a test tube, and shakensufficiently at 37° C. for 5 minutes. Then, an enzyme solution is addedthereto, and thus obtained reaction mixture is made up to 3 ml withammonium buffer solution. Reaction is conducted at 37° C. for 10 minuteswith shaking.

On the other hand, similar procedure is repeated using water in place ofthe test solution as a reference.

The OD value at 500 nm of the test solution is measured, and adifference ΔOD from the reference is 0.230. From the working curve ofFIG. 11, glycerol content in the test solution is determined to be 0.380mM.

What is claimed is:
 1. A method for the quantitative determination of glycerol in solution which comprises reacting glycerol oxidase, an enzyme having the ability to oxidize glycerol in the presence of oxygen to form hydrogen peroxide and gylceraldehyde, with the glycerol in the presence of oxygen and measuring the amount of oxygen consumed, or the amount of hydrogen peroxide or glyceraldehyde formed in an aqueous medium by the action of said glycerol oxidase, said amount being an indication of the concentration of glycerol in said solution.
 2. The method of claim 1, wherein the formed hydrogen peroxide is quantitatively determined by reacting the peroxide with 4-aminoantipyrine and phenol in the presence of peroxidase to obtain quinoneimine pigment, which is quantitatively determined by measuring the optical density of the reaction solution at 500 nm.
 3. The method of claim 1, wherein the glyceraldehyde formed is reacted with 2,4-dinitrophenylhydrazine, to form 2,4-dinitrophenylhydrazone and the 2,4-dinitrophenylhydrazone is quantitatively determined by colorimetry, this providing a quantitative determination of glycerol.
 4. The method of claim 1, wherein the amount of oxygen consumed is measured by an oxygen electrode and a Warburg's manometer. 